A striking feature of development in the Drosophila eye is the simultaneous synchronization of cell-cycle progression in G1 and the onset of pattern formation mediated by intercellular signaling molecules. rux mutants fail to arrest in G1, and instead all cells re-enter S phase precociously, resulting in defects in differentiation and cell fate determination . A genetic screen for loci that interact with rux in a dosage-sensitive fashion identified mutations in cyclin A (cycA) as a dominant suppressers of the rux mutant phenotype, suggesting that Rux functions to inhibit CycA expression and/or activity. We have shown that Rux physically interacts with CycA via a motif, RXL, that is conserved in a wide array of cyclin kinase inhibitors (CKIs) from yeast to mammals. In addition, Rux contains a C-terminal bipartite nuclear localization signal (NLS) that is required to target Rux to the nucleus in Drosophila tissue culture cells and in the developing eye. Mutation of the Rux NLS results in cytoplasmic localization of CycA, while in the eye mitosis is blocked and S phase is prolonged relative to wild-type. This suggests that nuclear localization of CycA is necessary for its S/G2 functions. Overexpression of Rux can drives CycA into the nucleus, where it is targeted for degradation by the anaphase-promoting complex/cyclosome (APC/C). Our recent studies of a mutation in the largest subunit of the APC/C, shattered (shtd), shows that cells in the mutant fail to arrest in G1 and accumulate CycA in a manner similar to that of rux mutants, consistent with a role for the APC/C in regulating this process. Further, Rux itself is destabilized in S phase cells that express the G1/S phase cyclin, Cyclin E (CycE). Rux degradation is dependent on the presence in the protein of four consensus sites for phosphorylation by cyclin-dependent kinases. We have demonstrated that Rux can bind CycE and is phosphorylated by CycE/Cdk complexes in vitro. These data support a model whereby Rux-mediated inhibition of CycA in the nucleus is relieved by CycE as cells re-enter S phase, releasing CycA for its S/G2 role. Current efforts in the lab are directed towards three overlapping and complementary areas. First, we are characterizing the mechanism by which Rux protein is degraded in S phase cells. We will pursue this directly, through an analysis of the interaction between CycE and Rux in vitro, and through the development of a tissue culture cell system to study CycE-dependent proteolysis of Rux protein. We will also characterize an eye-specific suppresser of the rux mutant phenotype, S(rux)2B, which may play a role in regulating CycE expression and/or activity. Second, we have characterized a mutation, shtd, in the largest subunit of the APC/C, APC1. We are using the shtd mutation to screen for genes that interact with the APC/C. In this manner, we hope to identify genes involved in regulating the APC/C during development, as well as targets for APC/C activity. Third, we have also identified the signaling molecules Hedgehog (Hh) and Patched (Ptc) as genetically interacting with rux. We are currently characterizing the pathway whereby Hh and Ptc regulate Rux activity. The high degree of conservation of cell cycle components between species makes it likely that many of the pathways for cell cycle regulation during development will be conserved during evolution.